The present invention relates to protein:phospholipid structures which are useful for stabilizing the secondary and tertiary structure of proteins capable of transitioning into the molten globular state. More particularly, this invention relates to G-CSF:phospholipid and MGDF:phospholipid compositions having increased stability, exhibiting increased shelf life, and capable of use in high temperature formulations and as novel delivery vehicles.
Several proteins have been shown to transition into a molten globular state (MGS). Van der Goot, F. G., Nature 354, 408-410 (1991). Proteins in the molten globular state exhibit secondary structure which is comparable to the native protein yet they lack rigid tertiary structure. Pitsyn et al., FEBS Letters 262:1, 20-24 (1990). In some cases, transition into this state is accompanied by exposure of previously hidden hydrophobic regions of the protein. By exposing critical hydrophobic residues, the MGS may be an intermediate in the aggregation and precipitation of proteins. The MGS conformation can be detected by comparing the circular dichroism in the far UV region with the spectra of aromatic side chains (near UV circular dichroism and fluorescence). The molten globular state exhibits aromatic group spectral changes in the absence of far UV circular dichroism changes, Bychkova et al., FEBS Letters 238: 231-234 (1988),and may be involved in membrane penetration by some proteins Bychkova et al., FEBS Letters 238: 231-234 (1988); Van der Goot, F. G., Nature 354, 408-410 (1991).
Granulocyte colony stimulating factor (G-CSF) is one protein known to transition into the MGS prior to aggregation. Human recombinant G-CSF selectively stimulates neutrophils, a type of white blood cell used for fighting infection. Currently, Filgrastim, a recombinant C-CSF, is available for therapeutic use. The structure of G-CSF under various conditions has been extensively studied; Lu et al., J. Biol. Chem. Vol. 267, 8770-8777 (1992). Because of its hydrophobic characteristics, G-CSF is difficult to formulate for extended shelf life. Formulations of certain hydrophobic proteins lose activity due to formation of dimer and higher order aggregates (macro range) during long-term storage. Other chemical changes, such as deamidation and oxidation may also occur upon storage. In addition, the G-CSF formulator must protect against denaturation and, in particular, look to maintain the stability of the secondary and tertiary structure of the protein.
Human GM-CSF is a 22-kDa glycoprotein required continuously for the in vitro proliferation of macrophage and granulocytic progenitor cells. It also controls the irreversible commitment of these progenitors to form granulocytes and macrophages. Other biological activities may include regulation of the functional activity of mature cell types; Gough et al., Nature, 309, 763-767 (1984), and increasing chemotaxis towards recognized chemoattractants; Williams et al., Hematology, 4th ed. (1990). GM-CSF also stimulates the production of monocytes, and thus may be useful in the treatment of monocytic disorders, such as monocytopenia.
Human GM-CSF can be obtained and purified from a number of sources. Procedures for the production of recombinant human GM-CSF have been previously described by Burgess et al., Blood, 69:1, 43-51 (1987). U.S. Pat. No. 5,047,504 (Boone), incorporated herein by reference, has enabled the production of commercial scale quantities of GM-CSF in non-glycosylated form as a product of procaryotic host cell expression.
MGDF, or megakaryocyte growth and differentiation factor, is a recently cloned cytokine that appears to be the major regulator of circulating platelet levels. See Bartley, T. D. et al., Cell 77:1117-1124 (1994); Lok, S. et al., Nature 369:565-568 (1994); de Sauvage, F. J. et al., Nature 369:533-538 (1994); Miyazake, H. et al., Exp. Hematol. 22:838 (1994); and Kuter, D. J. et al., PNAS USA, 91:11104-11108 (1994). MGDF is also referred to as thrombopoietin (TPO), mpl-ligand, and megapoietin. Mature human MGDF is a protein having 332 amino acids in total. The sequence of this protein and the corresponding cDNA are shown in FIG. 29 herein.
Recombinant MGDF produced in both Chinese Hamster Ovary (CHO) and E. coli cells has been demonstrated to have a biological activity of specifically stimulating or increasing megakaryocytes and/or platelets in vivo in mice, rats and monkeys. See e.g., Hunt, P. et al., Blood 84(10):390A (1994). Human MGDF molecules that have been truncated so that they extend at least 151 amino acids, starting from amino acid position 1 in FIG. 29, retain biological activity in vivo. It is also possible to remove up to the first six amino acids at the N-terminus of the human sequence MGDF protein and retain biological activity. Therefore, it appears that biological activity is retained within amino acids 7 to 151 (inclusive) of the mature amino acid sequence of human MGDF shown in FIG. 29.
Naturally occurring MGDF is a glycosylated molecule. The glycosylation pattern of natural MGDF is related to two key domains that have been found in MGDF. The sequence of the first approximately 151 amino acids of human MGDF, corresponding to an active portion of the molecule, bears notable homology to erythropoietin (EPO), a cytokine capable of stimulating production of erythrocytes, and is referred to as the "EPO-like" domain of human MGDF. The remaining amino acids of the mature protein make up a so-called "N-linked carbohydrate" domain, since they include most if not all of the sites for N-linked glycosylation. In human MGDF, there are six N-linked glycosylation sites all contained in the N-linked glycosylation domain. Both domains contain O-linked glycosylation sites. There are an estimated 12-14 O-linked glycosylation chains in the molecule. Experimental evidence with human MGDF DNA expressed recombinantly in CHO cells reveals that in the EPO-like domain at least two O-linked sites are glycosylated, at positions 1 (Ser) and 37 (Thr).
While proteins such as G-CSF and MGDF may be stabilized under certain defined conditions, there still exists a need to extend the shelf life of these and other proteins by stabilizing the secondary and tertiary structure of the proteins. One way which has been tried in the past to work with such proteins is the use of liposomes. Liposomes are completely closed lipid bilayer membranes formed by water-insoluble polar lipids, particularly phospholipids. Liposome vesicles may have a single membrane bilayer (unilamellar) or may have multiple membrane bilayers (multilamellar). The bilayer is composed of two lipid monolayers having a hydrophilic (polar) "head" region and a hydrophobic (nonpolar) "tail" region wherein the hydrophobic tails orient toward the center of the bilayer, whereas the hydrophilic heads orient toward the aqueous phase. The stability, rigidity, and permeability of liposomes can be altered by changes in the phospholipid composition, by changes in temperature, by inclusion of a sterol or by incorporation of a charged amphiphile. The basic structure of liposomes may be made by a variety of techniques known in the art.
In the process of their formation liposomes can entrap water solutes in the aqueous channels and release them at variable rates. Upon the discovery that liposomes can introduce enzymes into cells and alter their metabolism (Gregoriadis, New Engl. J. Med. 295, 704-710, 765-770 (1976)), liposomes were heralded as the answer to the quest for targeted drug delivery. As a result, there is a great deal of developmental research in the pharmaceutical industry involving use of liposomes as slow release depots for drugs, vitamins and proteins sequestered within the hydrophobic layers or hydrophobic core of the liposome.
Successful use of liposomes as drug-carriers has been limited because the researchers attempting such use have encountered several problems. For example, liposomes are known to act as powerful immunological adjuncts to entrapped antigens and caution must be exercised when enzymes or other proteins of xenogenic origin are entrapped in the liposomes. Also, the rate of diffusion of the drug is difficult to control. This is a function of the inherent instability of the liposomes and the presence of certain blood components which accelerate diffusion of certain drugs. In addition, by their nature, some substances are poorly entrapped in liposomes and therefore diffuse rapidly in circulation. Finally, there has been a problem targeting any cells or organ other than the liver or spleen. An excellent review of liposomes, substances which have been incorporated into liposomes, and the problems associated with use of liposomes as drug carriers is "Liposomes" by Gregory Gregoriaidis, found in Drug Carriers in Biology and Medicine, Chapter 14, 287-341 (Academic Press, N.Y., 1979).
While much has been reported concerning attempts to use liposomes as drug carriers, little has been disclosed concerning the use of liposomes for purposes of increasing shelf life of therapeutic peptides or proteins by stabilizing the structure of the peptide and/or protein. In PCT/US90/05163, entitled, "Therapeutic Peptides and Proteins", Hostetler, et al. disclose use of empty liposomes as a pharmaceutically acceptable diluent to solubilize polypeptides and/or proteins in order to prevent accumulation of the polypeptides and/or proteins at an air/water interface, and to prevent adsorption of the polypeptides and/or proteins to container surfaces. Hostetler et al. disclose that negatively charged phospholipid may be added up to about 50 mole percent, and that phosphatidylcholine, a neutral phospholipid, is the preferred liposome. Hostetler et al. do not disclose a diluent shown to stabilize the structure of a polypeptide and/or protein.
In PCT/US91/07694, entitled, "Preparation and Characterization of Liposomal Formulations of Tumor Necrosis Factor", Hung et al., a lipophilic modified tumor necrosis factor (TNF) molecule in association with the surface or encapsulated within a liposome is described. The liposomal lipophilic TNF molecules are reported to have enhanced in vivo stability. Stability referred to a decrease or a decreased tendency of the TNF-liposome to leak TNF into the system in vivo. The preferred liposomes were neutral lipids. Hung et al. do not disclose a TNF composition wherein the excipients have a stabilizing effect on the structure of the protein.
Nothing can be drawn from the literature concerning contacting a protein, e.g. G-CSF or MGDF, with a negatively charged lipid vesicle thereby directly stabilizing the protein against thermally-induced aggregation, denaturation, loss of activity, and unfolding of the secondary structure. The need exists for such compositions which provide the benefit of being useful in formulation procedures requiring high temperatures (e.g. incorporation of G-CSF and/or MGDF into polymers) as well as being used as novel delivery vehicles (e.g. oral administration of pegylated G-CSF). The present invention provides such compositions.